Biochemical and structural characterization of recombinant copper-metallothionein from Saccharomyces cerevisiae
Sayers, Zehra and Brouillon, Patricia and Svergun, Dimitri I. and Zielenkiewicz, Piotr and Koch, Michel H. J. (1999) Biochemical and structural characterization of recombinant copper-metallothionein from Saccharomyces cerevisiae. European Journal of Biochemistry, 262 (3). pp. 858-865. ISSN 0014-2956
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Official URL: http://dx.doi.org/10.1046/j.1432-1327.1999.00451.x
Methods were developed for large-scale purification of recombinant Cu-metallothionein (Cu-MT) for structural investigations and the determination of Cu-binding stoichiometry. Cu-MT of Saccharomyces cerevisiae overexpressed in Escherichia coli was purified using a procedure based on ion exchange and gel filtration chromatography followed by reversed-phase HPLC. The purified protein was fully characterized by electrophoresis, amino acid analysis, atomic absorption spectroscopy and elemental analysis, and was shown to contain 10 +/- 2 Cu(I) per molecule of protein. Small angle X-ray scattering measurements yielded a radius of gyration of 1.2 nm for the recombinant protein, indicating a more extended structure in solution than that derived from the recent NMR data [Peterson, C.W., Narula, S.S. & Armitage, I.A. (1996) FEES Lett. 379, 85-93].
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